Endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe as well as synthesis method and application thereof
A nitroreductase, fluorescent probe technology, applied in fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., to achieve the effects of high yield, high sensitivity and simple synthesis method
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[0044] Example 1 Synthesis of fluorescent probe.
[0045] In a 25 mL round-bottom flask, slowly add 1 mmol of ethylenediamine (compound 1) dropwise to toluene (5 mL) at 40 ℃, add 1.0 mmol of p-toluenesulfonyl chloride (compound 2) with sufficient stirring, and heat After refluxing for 3-4 h, cool to room temperature, add 5ml of 2M HCl aqueous solution to the reaction, remove the filter residue by suction filtration, neutralize the filtrate with NaOH until a large amount of white solid precipitates, filter under reduced pressure and vacuum dry the solid to obtain a solid N-(2-aminoethyl)-4-methylbensulfonamide (Compound 3), yield: 72%. The product was directly subjected to the next reaction without purification.
[0046] In a 100 mL round-bottom flask, add 243 mg of 4-nitronaphthalene dicarboxylate (compound 4) (1 mmol) and 20 mL of ethanol, and add 0.1 mL of triethylamine and 321 mg of N-(2-aminoethyl) with stirring. )-4-methylbenzenesulfonamide (compound 3) (1.5 mmol), then heat...
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[0048] Example 2 Fluorescence probe Na-ER-NTR absorption spectrum test
[0049] The fluorescent probe Na-ER-NTR in Example 1 was weighed and prepared with dimethyl sulfoxide (DMSO) to prepare a 1 mM mother liquor.
[0050] Take 25 μL of the probe mother solution and add them to 3 identical 5 mL volumetric flasks, add 225 μL of DMSO solution, and add reduced coenzyme I (NADH, final concentration 300 μM) to two of them. One of the two volumetric flasks with coenzyme was added with phosphate buffer solution of nitroreductase (final concentration 2.0 μg / mL), and the other was not added. The three volumetric flasks are all fixed to volume with phosphate buffer (0.01M PBS, pH = 7.4), reacted at 37 ℃ for 60 min, and then subjected to the absorption spectrum test. The wavelength is the abscissa and the absorbance is the ordinate. figure 2 . by figure 2 It can be seen that the absorption intensity of a single probe is very small, and a weak absorption peak appears at 360 nm. After addin...
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[0051] Example 3 Kinetic test of the interaction between fluorescent probe Na-ER-NTR and nitroreductase.
[0052] The fluorescent probe mother solution in Example 2 was diluted to 5 μM probe working solution for later use.
[0053] The nitroreductase was prepared with a concentration of 0, 0.5, 1.0, 1.5, 2.5, and 5.0 μg / mL in PBS with a pH of 7.4 and a concentration of 0.01 M.
[0054] The probe working solution was mixed with different concentrations of nitroreductase, the final concentration of NADH was 300μM, and then the fluorescence detection (λ ex = 440 nm, λ em = 545 nm), test every 3 minutes, test for 80 minutes, record the fluorescence intensity of each system over time, and establish a curve of fluorescence intensity over time, such as Figure 4 It is shown that the fluorescence intensity of the reaction system gradually increases, and tends to be stable after nearly 60 minutes, and the fluorescence intensity of the reaction system reaches a saturated state.
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